Bacterial diversity significantly reduces toward the late stages among filarial lymphedema patients in the Ahanta West District of Ghana: A cross‐sectional study

Abstract Background Lymphatic Filariasis (LF), a neglected tropical disease, has been speculated to be complicated by secondary bacteria, yet a systematic documentation of these bacterial populations is lacking. Thus, the primary focus of this study was to profile bacteria diversity in the progression of filarial lymphedema among LF individuals with or without wounds. Methods A cross‐sectional study design recruited 132 LF individuals presenting with lymphedema with or without wounds from eight communities in the Ahanta West District in the Western Region, Ghana. Swabs from the lymphedematous limbs, ulcers, pus, and cutaneous surfaces were cultured using standard culture‐based techniques. The culture isolates were subsequently profiled using Matrix‐assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry. Results Of the 132 LF participants recruited, 65% (85) had filarial lymphedema with no wounds. In total, 84% (235) of the bacterial isolates were identified. The remaining 16% (46) could not be identified with the method employed. Additionally, 129(55%) of the strains belonged to the phylum Firmicutes, while 61 (26%) and 45 (19%) represented Proteobacteria and Actinobacteria, respectively. Generally, irrespective of the samples type (i.e., wound sample and non‐wound samples), there was a sharp increase of bacteria diversity from Stages 1 to 3 and a drastic decrease in these numbers by Stage 4, followed by another surge and a gradual decline in the advanced stages of the disease. The Shannon Diversity Index and Equitability for participants with and without wounds were (3.482, 0.94) and (3.023, 0.75), respectively. Further, Staphylococcus haemolyticus and Escherichia coli showed resistance to tetracycline, chloramphenicol, and penicillin. Conclusion The present study reveals a sharp decline in bacterial load at the late stages of filarial lymphedema patients. In addition, we report an emerging antimicrobial resistance trend of S. haemolyticus and E. coli against commonly used antibiotics such as tetracycline, chloramphenicol, and penicillin in communities endemic for LF in the Ahanta West District, Ghana. This could pose a huge challenge to the management of the disease; particularly as current treatments are not quite effective against the infection.


Conclusion:
The present study reveals a sharp decline in bacterial load at the late stages of filarial lymphedema patients. In addition, we report an emerging antimicrobial resistance trend of S. haemolyticus and E. coli against commonly used antibiotics such as tetracycline, chloramphenicol, and penicillin in communities endemic for LF in the Ahanta West District, Ghana. This could pose a huge challenge to the management of the disease; particularly as current treatments are not quite effective against the infection.

| BACKGROUND
The human skin is regarded as the largest organ and mainly serves as a protective barrier against biological, mechanical, and physical agents and is home for many microbes. 1 Recent studies have suggested that these microbes (archaea, bacteria, fungi, and viruses) play crucial roles in maintaining a healthy balance of human physiology, and thus, any significant change in their composition may lead to a disease state. 2 For instance, scientific evidence suggests that a substantial change in the microbiota could possibly be the reason for many skin infections such as atopic dermatitis, psoriasis, hidradenitis suppurativa, and acne. 3 This knowledge has garnered the idea of manipulating microbial communities in treating illness. 4 Thus, the NIH Common Fund Human Microbiome Project (HMP) established in 2008 to characterize the human microbiome and analyze its role in human health and disease, has made substantial strides in decoding the exact functional roles of the human microbiome, particularly the skin microbiome in diseases. 5,6 Despite increasing evidence of the role of microbiota in diseases, there is a dearth of information on the role of the microbiome in neglected tropical diseases, especially diseases which affect the skin.
Lymphatic filariasis (LF) has been hypothesized to be complicated by secondary bacteria, which aggravate the occasional severe pain people living with the infection experience. [7][8][9] Moreover, bacteriological study on the skin biopsy, lymph node, lymph showed populations of Staphylococcus epidermis, S. hominis, Micrococcus species and luteus, Corynebacterium, Streptococcus acidominus which hitherto are sterile sites. 8 More specifically, there has been a report of direct link between the group A streptococci infection with acute dermatolymphangioadenitis (ADLA) as a higher tire of antistreptolysin O (ASO) was observed to positively correlate with ADLA experience by LF patients. 10 This observation has been suggested to be due to the Gram-positive bacteria (group A streptococci) invading the skin barrier into the peripheral blood. 11,12 LF is documented to infect nearly 120 million individuals worldwide with almost 40 million individuals currently suffering from varying degrees of disability and morbidity. 13 Presently, Filariasis Test Strip (FTS) 14 is used to diagnose LF after several years of using the thick smear technique to detect the presence of microfilariae. 15 Lymphedema is the main sequelae of the infection affecting about 2% of the population of LF endemic areas. 16 Lymphedema is estimated to affect 15 million persons living with the disease. 13 However, studies have attributed the progression of the lymphedema of the leg to recurrent episodes of ADLA and the presence of lesions with varying sizes. 9,17 Lesions or wounds of LF individuals have been observed to be located mainly on the interdigital part of the feet with few dotted around the lower extremities. 9 These wounds serve as the entry points of secondary bacterial (microbial infection); thus, complicating the morbidity of LF patients. 17 Entry lesions in filarial lymphedema individuals are wounds located on the skin of affected limb, which function as entries for pathogens, 18 but these lesions can also be located on limbs without lymphedema. These wounds are mostly chronic, which tend to promote the growth of fungi, thus exacerbating the ADLA episodes leading to excruciating pain in patients. Considering that the second objective of the Global Programme for Elimination of Filariasis (GPELF) is to alleviate pain and morbidity among LF patients. Despite speculating possible secondary bacteria complicating LF, systematic documentation of these bacterial populations is limited.
Additionally, there is a growing concern of antimicrobial resistance of microbes, particularly as there is limited data on surveillance of antimicrobial resistance in sub-Saharan Africa. 19,20 From our field observation of LF endemic communities in the Ahanta West District of Ghana, affected individuals routinely use antibiotics when they have "filarial attacks," and some use it topically on their wounds. Thus, profiling the antimicrobial resistance and susceptibilities of isolated bacteria from these LF individuals is crucial.
In this study, we used conventional culture methods augmented with Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) technology to understand bacteria diversity in the progression of filarial lymphedema among LF individuals with or without wounds.

| Study design
This study was cross-sectional carried out in eight (8) LF-endemic communities in the Ahanta West District, Ghana. Identification of microbial populations was performed as previously described. 21 The study communities were Akatakyi, Princess Town, Dixcove, Ampatano, Asemkow, Butre, Achowa, and Busia, which are LF hotspots located at the Western Region of Ghana ( Figure 1).

| Study participants
Individuals presenting with filarial lymphedema with or without wounds and had resided in the community for the past ten (10) years were potential participants for the study. Participants were screened by experienced filarial experts and registered for the study if they gave their informed consent after thorough explanation of the study protocols in the local dialects were recruited onto the study.

| Data collection
Structured questionnaires were used to obtain socio-demography information of the study participants. Information about the pathology, such as the stage of the leg (severity of the disease), location of the wound and wound appearance was also obtained.
Wound swab or the skin swab of the lymphedematous leg was collected. Here, those presenting with wounds had their wounds and their edges cleaned with sterile saline or water. The Levine method of sample collection was implored as previously described. 22 All samples collected under aseptic conditions (i.e., clean work surface, perform hand hygiene with soap and water, etc.) and kept in Amine transport medium (Copan Diagnostics Inc.) which were immediately transferred into a liquid nitrogen tank (−120°C) and transported to the Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) laboratory, Kumasi, for further laboratory analysis.

| Bacterial culturing
To determine the bacteria composition of the swab samples, the wound or skin swabs were plated on both Columbia nalidixic acid (CNA) agar supplemented with 5% Sheep and MacConkey agar (Mac).
Under aerobic and anaerobic conditions, the samples were cultured on the respective media and then incubated overnight at 35-37°C.
The pure culture isolates from CNA and Mac were preserved in cryotubes with brain heart infusion (BHI) medium and stored at −80°C for later identification.

| Bacterial identification
To identify the bacterial isolates in the swab samples, the culture

| Statistical analysis
The data obtained from the structured questionnaires were entered into Epi Info Version 7. Any result with a p value less than 0.05 was considered statistically significant.

| Demography of study participants
In total, 132 LF participants were recruited for the study from eight (8) communities in the Ahanta West District. Of these, 93 (70%) were females. The age range of the participants was 18-81 years, with a median of 48 years (Table 1)

| Bacterial strains identified among the study participants
In all, 281 pure bacterial isolates were cultured, of which 235 (84%) were identified using the MALDI-TOF identification platform. Of the 235 bacteria identified, the most occurring bacterial genus was   Figure 4) while 83 bacteria were isolated from the male study participants with S. epidermis being the common isolates ( Figure 5).
Moreover, 129 (55%) of all the bacterial strains identified belonged to the phylum Firmicutes, with the remaining proportions shared between the phyla Proteobacteria (61, 26%) and Actinobacteria (45, 19%). A one-way ANOVA indicated a significant statistical difference between the phyla of bacteria strains (p = 0.000; Table 3).

| The bacteria populations among study participants
To determine the various bacterium types that consist of the microbial population of the study participants, the bacteria typed F I G U R E 2 The leg staging of lymphedema presented by study participants (Leg staging according to ref. [25] F I G U R E 3 A Venn diagram of bacterial communities in the wound and non-wound samples ASIEDU ET AL.  observed lower bacteria numbers (non-wound sample: 1; wound sample: 4). In addition, the phylum representations of bacteria strains among the various study participant's communities did not differ. As a trend of phylum Firmicutes was observed to be in higher numbers in most study communities followed by Proteobacteria and then Actinobacteria ( Figure 8).

| Antimicrobial susceptibility testing among bacteria strains in wound sample
Twenty-three (23) different bacteria genera were identified as Gram-positive bacteria from the wound samples. Seven (7) of the Gram-positive species showed an average of 60% resistance against nine (9) different antibiotics, with Staphylococcus haemolyticus showing the highest resistance (82%), followed by Brevundimonas diminuta, Globicatella sanguinis, and Corynebacterium spp., all revealing an average resistance of 78% to the antibiotics ( Figure 9). Moreover, the highest frequency of resistance was found against tetracycline (77%), penicillin (76%), and chloramphenicol (71%) among the 78 Gram-positive bacteria identified from the wound samples (Table 4).
Likewise, 21 different Gram-negative genera were observed in the wound samples, with 4 showing an average resistance of >60% against 11 antibiotics. E. coli showed an average of 76% resistance against the 11 antibiotics, followed by P. aeruginosa (68%), Wohlfahrtiimonas chitiniclastica (64%), and Acinetobacter baumannii (61%; Figure 10). Furthermore, chloramphenicol was observed to be the most resistant to the 44 Gram-negative bacteria identified in the wound samples (Table 4).

| Antimicrobial susceptibility testing among bacteria strains in non-wound sample
In all, 74 Gram-positive bacteria were identified in the nonwound samples and screened for antimicrobial susceptibility testing against nine different antibiotics. The results showed that four different bacteria species (i.e., Enterococcus raffinosus, S.

haemolyticus, Staphylococcus sciuri, and Streptococcus pyogenes)
showed an average resistance (>60%) against the antibiotics ( Figure 11). However, chloramphenicol (73%) and tetracycline (73%) were observed to be the common antibiotics that the Gram-positive strains were resistant to in the non-wound samples followed by penicillin (69%). It is also worth noting that Gram-positive bacteria in the non-wound samples were more sensitive to ciprofloxacin (69%) than any other antibiotic (Table 4).
In addition, 13 Gram-negative bacteria were identified among the non-wound sample type from 10 different genera. Burkholderia cenocepacia and A. baumannii were the only Gram-negative that showed an average resistance of >60% to the 11 antibiotics ( Figure 12). 77% of Gram-negative bacteria were resistant to ampicillin, followed by amoxicillin (69%) and tetracycline (69%).
T A B L E 3 A one-way analysis of variance of the bacteria phylum     These reports could affect the treatment of wounds with antibiotics as a significant proportion of isolates from wound cultures has been observed to show resistance to first-line antimicrobials in sub-Saharan Africa. 19,44 These resistance patterns may be community-acquired rather than hospital-acquired as most study participants hardly seek medical attention. This is partly due to the long distance of hospitals from the endemic communities and poor education on antibiotic use. The study participants usually resort to the local licensed chemical store for medication compared to the community health centers to cut down the cost of treatment and alleviate them from painful filarial attacks. 45 Elsewhere, the sensitivity trends among Gram-negative bacteria corroborate with this present study's finding as most of the Gram-negative bacteria were sensitive to ceftazidime, ciprofloxacin, gentamicin, cefuroxime, and clindamycin as well-documented. 19,38,[46][47][48] The main limitation of this study is the possible underestimation of the total bacterial diversity among the study participants as the traditional culture method is biased toward only a few bacteria species that are "culturable" on artificial culture media. Additionally, the study was restricted to only bacteria species; thus, further studies could be conducted to understand the dynamics of other microbes such as fungi, viruses among people living with filarial lymphedema. Also, future prospects can possibly take a closer look at how "good" bacteria community could be essential in reversing the severity of filarial lymphedema. Despite these limitations, our data show a significant reduction of bacteria diversity toward the advanced stages of filarial lymphedema among individuals living with LF in the Ahanta West District in Ghana, indicating that, based on our data, a selective predominance of a few bacteria, which suppress the growth of other bacteria.